1. Field of the Invention
The invention relates to a rapid and highly efficient method of labeling liposomes and liposome-encapsulated protein. In particular, the method relates to radionuclide labeling of preformed liposomes with or without encapsulated protein by means of a radionuclide carrier characterized as being membrane diffusible.
Table 1 is a list of abbreviations used.
TABLE 1 ______________________________________ cpm counts per minute DTPA diethylenetriaminepenta-acetic acid GBq gigabequerels HMPAO hexamethylenepropylene amine oxime LEH liposome-encapsulated hemoglobin PBS phosphate buffered saline PYP pyrophosphate Tc Technetium LUV large unilamellar vesicles ______________________________________
2. Description of Related Art
Liposomes are of considerable interest because of their value as carriers for diagnostic agents, particularly radiopharmaceuticals for tracer and imaging studies. Successful biodistribution studies, for example, require attachment of a radiolabel to the liposome. Unfortunately, the entrapment of water soluble radionuclides within the liposome is relatively inefficient. Another major problem in using liposomes is their leakiness, resulting in limited usefulness for many applications (Hwang, K. J., in Liposomes from Biophysics to Therapeutics, M. J. Ostru, Ed., Marcel Dekker, N.Y., 1987).
Radioactive markers have been widely used as a non-invasive method for studying the distribution of drugs in vivo. The use of gamma emitting radioisotopes is particularly advantageous because, unlike beta-emitters, they can easily be counted in a scintillation well counter and do not require tissue homogenization prior to counting. In addition, gamma-emitters can be imaged with nuclear gamma cameras. With this type of imaging, the dynamic biodistribution can be followed non-invasively using consecutive one minute computer acquired scintigraphic images which are analyzed to calculate organ biodistribution curves.
The most common radiolabel used in diagnostic radiopharmaceuticals today is .sup.99m Tc. This radionuclide is produced from the beta decay of .sup.99 molybdenum and has a half-life of 6 hours. It is widely available from a generator system at low cost and its relatively short half-life provides for safer and more convenient handling than other available radionuclides. Its gamma emission is in the range of 140 Kev which is an ideal range for producing high resolution images (Caride, V. J. and Sostman, H. D. in Liposome Technology, Vol. II, G. Gregoriadis, Ed., CRC Press, Boca Raton, 1984). Heptavalent .sup.99m TcO.sub.4.sup.- is produced from the generator and since it is relatively unreactive, must be reduced to a lower oxidation state before use as a radiopharmaceutical. Stannous chloride is the most commonly used reducing agent (Barratt, G. M., Tuzel, N. S. and Ryman, B. E. in Liposome Technology, Vol. II, G. Gregoriadis, Ed., CRC Press, Boca Raton, 1984).
Radiolabeled complexes have been employed as a means for labeling liposomes. Isonitrile radionuclide complexes of Tc and other gamma-emitters appear to have use for labeling vesicles with lipid membranes, including red blood cells (U.S. Pat. No. 4,452,774, Jones et al., Jun. 5, 1984). Propylene amine oxime complexes with .sup.99m Tc are stable neutral lipophilic complexes which have been approved for radioimaging in vivo as an adjunct in the detection of altered regional cerebral perfusion (Ceretec.TM.). These complexes which diffuse across cellular walls have been shown to localize in red blood cells, although radioactivity is readily washed from the cells. (U.S. Pat. No. 4,789,736, Canning et al., Dec. 6, 1988 and U.S. Pat. No. 4,615,876, Troutner et al., Oct. 7, 1986). Furthermore, the usefulness of these complexes is limited because the complexes are not stable. Ceretec.TM., for example, has a useful life of approximately 30 minutes.
The radionuclide of .sup.111 indium (.sup.111 In) has found some use as an imaging agent. Multilamellar lipid vesicles labeled with .sup.111 In using 8-hydroxyquinoline showed a labeling efficiency of 30% (Caride, V. J. and Sostman, H. D. in Liposome Technology, Vol. II, G. Gregoriadis, Ed., CRC Press, Boca Raton, 1984). Higher labeling efficiencies have been shown for loading .sup.111 In into the aqueous compartment of liposomes. Acetylacetone, a water soluble lipophilic chelator, can be complexed with .sup.111 In. This is then mixed with liposome-encapsulated nitrilotriacetic acid with subsequent formation of labeled nitrilotriacetic acid. The resulting labeled liposomes are unstable unless excess acetylacetone is removed by an ion exchange process (Beaumier, P. L. and Hwang, K. J., J. Nucl. Med., 23, 810-815 (1982)).
In general, labeling efficiency of 50-70% for .sup.99m Tc has been reported for multilamellar vesicles and 4-20% for small unilamellar vesicles when using stannous chloride to reduce the pertechnetate. A persistent problem in all these methods is the removal of excess reducing agent as well as elimination of free pertechnetate. Separation can be done by gel filtration or dialysis, but there is often formation of a .sup.99m Tc-tin chloride colloid which is not readily distinguishable or separable from the liposomes (Barratt, G. M., Tuzel, N. S. and Ryman, B. E. in Liposome Technology, Vol. II, G. Gregoriadis, Ed., CRC Press, Boca Raton, 1984). This confounds the results of biodistribution studies since interpretation may be subject to altered uptake influenced by the labeled colloidal tin.
Attempts at labeling liposomes with imaging radiotracers have produced variable results (Barratt, G. M., Tuzel, N. S. and Ruman, B. E. in Liposome Technology, Vol. II, G. Gregoriadis, Ed., CRC Press, Boca Raton, 1984; Caride, V. J. and Sostman, H. D. in Lipid Technology, Vol. II, G. Gregoriadis, Ed., CRC Press, Boca Raton, 1984; Caride, V. J., Nucl. Med. Biol., 17, 35-39 (1990); Hwang, K. J. in Liposomes from Biophysics to Therapeutics, M. J. Ostro, Ed., Marcel Dekker, Inc., N.Y., 1987). Many radioisotope labels weakly bind to liposomes resulting in inaccurate biodistribution data. A more efficient imaging label procedure uses .sup.111 indium chloride (.sup.111 InCl) and nitrilotriacetic acid, a metal chelator (Beaumier, P. L. and Hwang, K. J., J. Nucl. Med., 23, 810-815 (1982); Turner, A. F., Presant, C. A., Proffitt, R. T., Williams, L. E., Winsor, D. W., Werner, J. L., Radiology, 166, 761-765 (1988); Proffitt, R. T., Williams, L. E., Presant, C. A., Tin, G. W., Uliana, J. A., Gamble, R. C. and Baldeschwieler, J. D., J. Nucl. Med., 24, 45-51 (1983). The nitrilotriacetic acid is incorporated into the liposome during the manufacturing process. The preformed liposomes are then incubated for 30 minutes with .sup.111 InCl. Although the .sup.111 InCl nitrilotriacetic acid labeling method has proven to be effective and the label tightly attached to the liposome, a heating step (60.degree. C.) is required, which adds to the time and inconvenience involved in the preparation. In a clinical situation convenience and speed are important. A further consideration is the expense of the .sup.111 In radionuclide. The present cost of .sup.111 In is approximately $135/mCi while cost of .sup.99m Tc, a superior imaging agent, is $0.35/mCi. This difference is highly significant in determining cost of imaging procedures to the patient and in a decision by the health provider to offer such services.
Other labeling carriers have been tried. Small amounts of octadecylamine-DTPA in liposomes have been shown to rapidly label the liposomes with .sup.67 Ga or .sup.99m Tc by chelation with efficient labeling, but over 30% of the label is lost after a 2 hour incubation in plasma (Hnatowich, D. J., Friedman, B., Clancy, and Novak, M. J. Nucl. Med., 22, 810-814 (1981).
The reasons for instability of .sup.99m Tc labeled liposomes are not well understood, although instability may be related to the liposome surface charge. Recent work has shown that the in vitro methods currently used to assess the stability of labeled liposomes do not predict isotope stability in vivo, and that the nature of the binding between the isotope and the liposome surface is important in regulating in vivo isotope stability (Love, W. G., Amos, N., Williams, B. D., and Kellaway, I. W., J. Microencapsulation, 6, 103-113 (1989)). The result is that even when labeling methods appeared to be highly efficient, and little instability was demonstrated in plasma or serum, significant loss of label could occur when the labeled liposomes were introduced into an animal or human.
Despite attempts to develop stable .sup.99m Tc-labeled liposomes, there has been little success. In a thoroughly detailed review of liposomal labeling with radioactive technetium, Barratt et al. noted that technetium labeling techniques vary widely in efficiency. Moreover, stability is generally recognized to be poor, especially in vivo. Most methods of labeling liposomes with .sup.99m Tc encapsulate the .sup.99m Tc during liposome manufacture. However, these encapsulation methods do not solve the problem of in vivo dissociation of .sup.99m Tc from the liposome. The dissociated .sup.99m Tc is usually visualized in the kidneys and bladder. These problems clearly illustrate that development of a reliable method to load high levels of .sup.99m Tc into liposomes without in vivo dissociation would be beneficial in view of the many clinical uses for radiolabeled liposomes (Hwang, K. J. in Liposomes from Biophysics to Therapeutics, M. J. Ostro, Ed., Marcel Dekker, N.Y., 1987).
There are numerous clinical applications for .sup.99m Tc-liposomes. Comparison studies of liposome scanning, bone scanning and radiography have been performed in inflammatory joint disease. Liposome scans have been shown to be positive only in clinically active inflammatory disease. The method has also been able to discriminate between different grades of joint tenderness, in contrast to bone scans (O'Sullivan, M. M., Powell, N., French, A. P., Williams, K. E., Morgan, J. R., and Williams, B. D., Ann. Rheum. Dis., 47, 485-491, 1988; Williams, B. D., O'Sullivan, M. M., Saggu, G. S., et al., Ann. Rheum. Dis. (UK), 46, 314-318 (1987)). Other studies include the localization of abscesses (Morgan, J. R., Williams, K. E., Davies, R. L., et al., J. Med. Microbiol., 14, 213-217 (1981); tumor scanning (Eisenhut, M., Therapiewoche (West Germany) 30, 3319-3325 (1980); lymph node imaging (Osborne, M. P., Richardson, V. J., Jeyasingh, K., Ryman, B. E., Int. J. Nucl. Med. Biol. (England) 6, 75-83 (1979; Yu, B., Chin. J. Oncol. (China) 10, 270-273 (1988); clearance in the human lung (Farr, S. J., Kellaway, I. W., Parry-Jones, D. R., Woolfrey, S. G., Int. J. Pharm. (Netherlands) 26, 303-316 (1985)); and infarction (Palmer, T. N. Caride, V. J., Caldecourt, M. A., Twickler, J., and Abdullah, V., Biochim. Biophys. Acta 797, 363-368 (1984)).
Other potential uses of a liposome label include cardiac gated blood pool angiography and gastrointestinal bleeding detection. The most commonly used process known as the modified in vivo technique is fairly lengthy and requires 2-3 injections into the patient. For red blood cell labeling, the patient is injected with 1-2 mg of stannous PYP (Callahan, R. J., et al., J. Nuclear Medicine 23, 315-318 (1982)). Fifteen minutes later a blood sample is withdrawn and incubated with .sup.99m TcO.sub.4.sup.- (free pertechnetate). The patient is then reinjected with the radiolabeled blood, the whole procedure requiring up to 1 hour. The major disadvantage of this technique is that the label is often poor and free pertechnetate is taken up in the stomach, resulting in intestinal contamination and making the results difficult to interpret. A rapid labeling technique would very likely alleviate this major problem, allowing improved cardiac and gastrointestinal bleeding detection imaging.
There is a distinct need for radiopharmaceutical materials that can be broadly applied to clinical applications and to biodistribution and bioimaging studies. .sup.99m Tc labeled liposomes would appear to be an ideal reagent but present methods of labeling liposomes with .sup.99m mTc are generally inefficient. A far greater problem is the lack of in vivo stability of .sup.99m Tc labeled liposomes, thereby limiting their use and creating uncertainty in interpretation of results.
The present invention is the surprising discovery that incubation of encapsulated reducing agent with liposomes, radionuclide labeled liposomes having high in vivo stability can be readily and efficiently prepared. The liposomes, preferably labeled with .sup.99m Tc, are useful in a wide range of clinical applications related to biodistribution and imaging. Labeled liposome-encapsulated protein may also be prepared by this method and has also been shown to have high stability in vivo.